WO2010099318A1 - Préparation d'échantillon physiologique pour nanodétecteurs - Google Patents

Préparation d'échantillon physiologique pour nanodétecteurs Download PDF

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Publication number
WO2010099318A1
WO2010099318A1 PCT/US2010/025412 US2010025412W WO2010099318A1 WO 2010099318 A1 WO2010099318 A1 WO 2010099318A1 US 2010025412 W US2010025412 W US 2010025412W WO 2010099318 A1 WO2010099318 A1 WO 2010099318A1
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chip
microfluidic purification
purification chip
biomarker
microfluidic
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PCT/US2010/025412
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English (en)
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Tarek M. Fahmy
Eric D. Stern
Mark A. Reed
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Yale University
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Publication of WO2010099318A1 publication Critical patent/WO2010099318A1/fr
Priority to US13/218,846 priority Critical patent/US9739771B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4725—Mucins, e.g. human intestinal mucin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

  • a biomarker is a term used to refer to a biomolecule or cell that may be measured in the blood or tissue of an individual, and which concentration reflects the presence or severity of a disease state in the individual.
  • Biomarkers may be specific cells, molecules, genes, gene products, proteins, enzymes, or hormones. Complex organ functions or general characteristic changes in biological structures may also serve as biomarkers.
  • a biomarker may be used as an indicator of the biological or metabolic state of an organism. More specifically, changes in the amounts of a biomarker in an individual may be correlated with the progression of a disease in the individual, the risk of the individual to develop a disease, or the susceptibility of a disease in the individual to a given treatment.
  • Biomarkers have emerged as potentially important diagnostic tools for cancer and many other diseases. Continuing discoveries of such biomarkers and their aggregation into molecular signatures suggests that multiple biomarkers may be necessary to precisely define disease states. Parallel detection of biomarker arrays is thus essential for translation from benchtop discovery to clinical validation. Such a technique would enable rapid, point-of-care applications requiring immediate diagnosis from a physiological sample. Critically, such a system should also be capable of detecting very low levels of aberrant genes and proteins, as many biomarkers are present at minute concentrations during early disease phases (Etzioni et al., 2003, Nature Rev. Cancer 3:243-252; Liang & Chan, 2007, Clin. Chim.
  • the invention includes a microfluidic purification chip for capturing at least one biomarker from a physiological solution.
  • the microfluidic purification chip comprises an immobilized first antibody directed to the at least one biomarker.
  • the immobilized first antibody is attached to the microfluidic purification chip by a molecular crosslinker.
  • the molecular crosslinker comprises a molecular spacer and a cleavable group.
  • the at least one biomarker is PSA (SEQ ID NO: 1) or CA15.3 (SEQ ID NO:2).
  • the molecular spacer is selected from the group consisting of a peptide, a nucleic acid, a polyethylene glycol, and an alkylene group.
  • the molecular spacer is a nucleic acid.
  • the nucleic acid is a single-stranded DNA oligonucleotide.
  • the nucleic acid is 5'-CGT AGA GGT TCA GTT GCA GC-3' (SEQ ID NO:3).
  • the cleavable group is a photocleavable group. In another embodiment, the group is l-(4'-amino-2'-nitro-phenyl)ethyl.
  • the molecular crosslinker further comprises a biotin-containing moiety. In another embodiment, the molecular crosslinker is Compound (I):
  • the amino group in Compound (I) is coupled through an amide bond to a carboxylic acid of the first antibody.
  • the microfluidic purification chip is derivatized with avidin and the biotin-containing moiety of the molecular crosslinker binds to the avidin.
  • the pillars in the microfluidic purification chip are about 100 ⁇ m in diameter. In another embodiment, the number of rows of the pillars ranges from about 25 to about 40. In yet another embodiment, the number of rows of the pillars is about 30. In yet another embodiment, the pillars in the microfluidic purification chip are arranged in a hexagonal geometry. In yet another embodiment, the width of the microfluidic purification chip ranges from about 2 mm to about 6 mm. In yet another embodiment, the width of the microfluidic purification chip is about 4 mm. In yet another embodiment, the length of the microfluidic purification chip ranges from about 5 mm to about 10 mm.
  • the length of the microfluidic purification chip is about 7 mm. In yet another embodiment, the height of the microfluidic purification chip ranges from about 50 ⁇ m to about 200 ⁇ m. In yet another embodiment, the height of the microfluidic purification chip is about 100 ⁇ m in height.
  • the microfluidic purification chip is connected by a duct to a sensing chip, whereby solution flows from the microfluidic purification chip to the sensing chip through the duct.
  • the duct optionally comprises a valve.
  • the maximum volume of solution held in the microfluidic purification chip is about half of the maximum volume of solution held in the sensing chip. In yet another embodiment, the maximum volume of solution held in the microfluidic purification chip is about 5 ⁇ L.
  • the sensing chip is derivatized with a second antibody directed to the at least one biomarker.
  • the sensing chip is a nanoribbon sensor.
  • the gate voltage (FQ) for the sensing chip ranges from about -2.5 V to about -6 V. In yet another embodiment, the gate voltage (F G ) for the sensing chip is about -5 V.
  • the invention includes a method of capturing and releasing at least one biomarker, wherein the at least one biomarker is originally in a physiological solution.
  • the method comprises the step of contacting the physiological solution comprising the at least one biomarker with a microfluidic purification chip, wherein the microfluidic purification chip comprises an immobilized first antibody directed to the at least one biomarker.
  • the immobilized first antibody is attached to said microfluidic purification chip by a molecular crosslinker, and the molecular crosslinker comprises a molecular spacer and a cleavable group.
  • the method further comprises removing the physiological solution from the microfluidic purification chip.
  • the method further comprises optionally washing the microfluidic purification chip with a buffer.
  • the method further comprises cleaving the cleavable group in a sensing buffer, to generate a biomarker- containing solution.
  • the at least one biomarker is PSA (SEQ ID NO:1) or CAl 5.3 (SEQ ID NO:2).
  • the molecular spacer is selected from the group consisting of a peptide, a nucleic acid, a polyethylene glycol, and an alkylene group. In another embodiment, the molecular spacer is a nucleic acid. In yet another embodiment, the nucleic acid is a single-stranded DNA oligonucleotide. In yet another embodiment, the nucleic acid is 5 '-CGT AGA GGT TCA GTT GCA GC-3 ' (SEQ ID NO: 1).
  • the cleavage is performed with UV or visible light and the cleavable group is a photocleavable group.
  • the photocleavable group is l-(4'-am,ino-2'-nitro-phenyl)ethyl.
  • the molecular crosslinker further comprises a biotin-containing moiety.
  • the molecular crosslinker is Compound (I):
  • the primary amino group in Compound (I) is coupled through an amide bond to a carboxylic acid of the first antibody.
  • the microfluidic purification chip is derivatized with avidin and the biotin-containing moiety of said molecular crosslinker binds to the avidin.
  • the pillars in the microfluidic purification chip are about 100 ⁇ m in diameter. In another embodiment, the number of rows of pillars in the microfluidic purification chip ranges from about 25 to about 40. In yet another embodiment, the pillars in the microfluidic purification chip are arranged in a hexagonal geometry. In yet another embodiment, the width of the microfluidic purification chip is about 4 mm. In yet another embodiment, the length of the microfluidic purification chip is about 7 mm. In another embodiment, the height of the microfluidic purification chip is about 100 ⁇ m.
  • the invention includes a method of pre-purifying and measuring the concentration of at least one biomarker in a physiological solution.
  • the method comprises the step of contacting the physiological solution comprising the at least one biomarker with a microfluidic purification chip, wherein the microfluidic purification chip comprises an immobilized first antibody directed to the at least one biomarker.
  • the immobilized first antibody is attached to the microfluidic purification chip by a molecular crosslinker, and the molecular crosslinker comprises a molecular spacer and a cleavable group.
  • the method further comprises the step of removing the physiological solution from the microfluidic purification chip.
  • the method further comprises the step of optionally washing the microfluidic purification chip with a buffer.
  • the method further comprises the step of cleaving the cleavable group in a sensing buffer, to generate a biomarker-containing solution.
  • the method further comprises the step of transferring the biomarker-containing solution to a sensing chip, wherein the sensing chip is derivatized with a second antibody directed to the at least one biomarker.
  • the method further comprises the step of contacting the biomarker-containing solution with the sensing chip.
  • the method further comprises the step of determining concentration of the biomarker in the biomarker-containing solution.
  • the method further comprises the step of determining concentration of the biomarker in the physiological solution.
  • the at least one biomarker is PSA (SEQ ID NO:1) or CA15.3 (SEQ ID NO:2).
  • the microfluidic purification chip is connected to the sensing chip by a duct, wherein the duct is used to transfer said biomarker- containing solution from the microfluidic purification chip to the sensing chip.
  • the duct optionally comprises a valve.
  • the maximum volume of solution held in the microfluidic purification chip is about half of the maximum volume of solution held in the sensing chip.
  • the maximum volume of solution held in the microfluidic purification chip is about 5 ⁇ L.
  • the sensing chip is a nanoribbon sensor.
  • the gate voltage (F G ) for the sensing chip ranges from about -2.5 V to about -6 V.
  • the gate voltage (VQ) for the sensing chip is about -5 V.
  • the sensing solution is 1 mM bicarbonate buffer.
  • Figure 1 is a schematic illustration of the operation of a microfluidic purification chip.
  • Figure IA illustrates first antibodies to multiple biomarkers (in a non-limiting example, prostate specific antigen - PSA - and carbohydrate antigen-15.3 - CAl 5.3) bound with a photocleavable crosslinker to the microfluidic purification chip.
  • the chip is contained in a plastic housing and a valve directs flow exiting the chip to either a waste receptacle or the nanosensor chip.
  • Figure 2 is a series of images illustrating the modeling results for biomarker binding in chips.
  • Figure 2A illustrates the velocity profile, in term of streamlines, for a flow rate of 10 ⁇ L/min.
  • Figure 2B illustrates the velocity profile, in term of velocities, for a flow rate of 10 ⁇ L/min.
  • Figure 2C illustrates the PSA retention for a flow rate of 10 ⁇ L/min.
  • Figure 2D illustrates the PSA retention for a flow rate of 1 ⁇ L/min.
  • Figure 3 is a series of images illustrating the operation of the microfluidic purification chip.
  • Figure 3 A illustrates the molecular structure of a photocleavable crosslinker contemplated within the invention. First- antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left).
  • Figure 3 C is a schematic representation of PSA and CA 15.3 detection using a modified ELISA technique.
  • Figure 3D illustrates the fluorescence optical micrograph of an anti-OVA functionalized microfiuidic purification chip following OVA-FITC-spiked whole blood flow and washing.
  • the inset plots the pixel intensity (iii) (determined by ImageJ) versus position for the cut line (shown as a broken line) (i) and similar cutlines from images of post-UV irradiation and transfer (ii) and of an anti-PSA functionalized microfiuidic purification chip following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images.
  • Figure 3E is a scatter plot showing the concentration of PSA released from the capture-release (cap- rel) chip versus the concentration of PSA introduced in whole blood.
  • Figure 3F is a scatter plot showing the concentration of CAl 5.3 released from the capture-release (cap-rel) chip versus the concentration of CAl 5.3 introduced in whole blood.
  • each datapoint represents the average of three separate microfiuidic purification chip runs and error bars represent one standard deviation.
  • Figure 4 is a schematic representation of the processing steps performed on standard one-side polished silicon wafers.
  • Figure 5 is a schematic representation of a chip assembly.
  • Figure 6 is a bar graph summarizing the immunoactivity determination of PSA and CA 15.3 by ELISA assays after UV irradiation.
  • Figure 7 is a schematic representation of the fabrication of a nanoribon sensor.
  • Figure 8 comprising Figures 8 A-8D, is a series of images representing optical micrographs of nanoribbon sensors.
  • Figure 8C illustrates the expansion of the region encompassed by the broken line box in Figure 8B.
  • Figure 8D illustrates the expansion of the region encompassed by the broken line box in Figure 8C.
  • Figure 9 is a series of images illustrating electrical characteristics of nanosensors.
  • Figure 9A is an optical image of devices outfitted with sensing reservoirs. The inset shows an optical micrograph of a completed device. Only the central region of the device (black arrow) is exposed to the solution. Metal leads contact the device source and drain and fan out to larger contacts (not shown). The 25 nm thick silicon device appears light gray.
  • Figure 9B is a / DS (F DS ) graph for VQ varied from 0 to -20V (arrow shows direction of increasing negative VQ) for a representative device illustrating p-type accumulation mode behavior.
  • Figure 10 comprising Figures 10A- 1OC, is a series of graphs illustrating the correlation of /with F G .
  • Figure 1OA illustrates the anti-PSA functionalized device backgating (using the handle wafer).
  • Figure 1OB illustrates the anti-CA15.3 functionalized device backgating (using the handle wafer).
  • Figure 1OC illustrates the solution gating.
  • Figure 11 is a series of graphs illustrating the correlation of / DS with F.
  • Figure 1 IA illustrates the correlation of / DS and F DS post- APTS functionalization.
  • Figure 1 IB illustrates the correlation of / DS and F G post- APTS functionalization.
  • Figure 11C illustrates the correlation of / DS and F DS after complete functionalization and FBS blocking.
  • Figure 1 ID illustrates the correlation of / DS and F G after complete functionalization and FBS blocking.
  • Figure 12 is a series of graphs illustrating the response of an anti-PSA functionalizing device to CAl 5.3 ( Figure 12A) and of an anti-CA15.3 functionalized device to PSA ( Figure 12B) in sensing buffer.
  • Figure 13 is a series of graphs illustrating absolute sensor responses.
  • Figure 13 A illustrates the absolute sensor response (not normalized) of anti-PSA functionalized device to the spiked buffer solution (top trace) or to the microfluidic purification chip-purified sensing experiments (bottom trace), as displayed in Figures 14A-14B.
  • Figure 13B illustrates the absolute sensor response (not normalized) of anti-CA15.3 functionalized device to the spiked buffer solution (top trace) or to the microfluidic purification chip-purified sensing experiments (bottom trace), as displayed in Figures 14A-14B.
  • Figure 13C illustrates the absolute sensor response for the unspiked control blood sample for the anti-PSA functionalize device, as displayed in Figures 14A-14B.
  • Figure 13D illustrates the absolute sensor response for the unspiked control blood sample for the anti-CA15.3 functionalize device, as displayed in Figures 14A-14B.
  • Figure 13E illustrates the absolute sensor response for the microfluidic purification chip-purified PSA-spiked blood sample for the anti-PSA functionalize device, as displayed in Figures 14E-14F.
  • Figure 13F illustrates the absolute sensor response for the microfluidic purification chip-purified CA15.3-spiked blood sample for the anti- CAl 5.3 functionalize device, as displayed in Figures 14E-14F.
  • Figure 14A illustrates response of an anti-PSA functionalized sensor to a microfluidic purification chip-purified blood sample initially containing 2.5 ng/ml PSA (and also 30 U/ml CAl 5.3), marked as (ii), or a control sample containing neither, marked as (i).
  • Figure 14B illustrates response of an anti-CA15.3 functionalized sensor to a microfluidic purification chip-purified blood sample initially containing 30 U/ml CA15.3 (and also 2.5 ng/ml PSA), marked as (ii), or a control sample containing neither, marked as (i).
  • Figures 14C and 14D illustrate the normalized response of two anti-PSA ( Figure 14C) and two anti-CA15.3 ( Figure 14D) functionalized devices to microfluidic purification chip-purified blood containing both PSA and CA15.3, with concentrations labeled. A least-squares fit is represented by a solid black line over the selected region (line endpoints).
  • the present invention relates to the discovery that a microfluidic purification chip may be used to pre-purify at least one biomarker of interest from a biological sample.
  • This microfluidic purification chip captures at least one biomarker from a solution, such a physiological solution. After washing of the chip, the at least one biomarker may be released into a solution suitable for detection and quantitation ofthe biomarker.
  • the invention provides a microfluidic purification chip for capturing and releasing at least one biomarker from a biological sample.
  • the invention provides a method of capturing and releasing at least one biomarker from a biological sample using a microfluidic purification chip.
  • the invention provides a method of measuring the concentration of a biomarker in a physiological solution, using a microfluidic purification chip for capturing and releasing the biomarker.
  • the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • physiological solution refers to any solution comprising physiological material isolated from a living organism.
  • physiological materials contemplated within the invention are blood, blood subtractions, serum, lymphatic fluid, saliva, urine, sweat, vaginal fluid and sperm.
  • the physiological solution comprises material selected from the group consisting of blood, blood subtractions, serum, lymphatic fluid, saliva, urine, sweat, vaginal fluid and sperm.
  • the physiological solution comprises blood.
  • PSA SEQ ID NO: 1 refers to prostate-specific antigen.
  • CA15.3 refers to Cancer Antigen 15-3.
  • polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • polypeptide sequences the left- hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl -terminus.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof.
  • a peptide that is not cyclic has an N-terminus and a C-terminus.
  • the N-terminus has an amino group, which may be free (i.e., as a NH 2 group) or appropriately protected (for example, with a BOC or a
  • the C-terminus has a carboxylic group, which may be free (i.e., as a
  • a cyclic peptide does not necessarily have free N- or C-termini, since they are covalently bonded through an amide bond to form the cyclic structure.
  • peptide bond means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated below: Full Name Three-Letter Code One-Letter Code
  • antibody refers to an immunoglobulin, whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These may be isolated from natural sources, or may be partly or wholly synthetically produced. Examples of antibodies are intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')2, Fv fragments, and single chain variable fragments (scFv), which are capable of binding an epitopic determinant.
  • Antibody fragments refer to antigen-binding immunoglobulin peptides that are at least about 5 to about 15 amino acids or more in length, and that retain some biological activity or immunological activity of an immunoglobulin.
  • Antibody as used herein includes polyclonal and monoclonal antibodies, hybrid, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library, and suitable derivatives.
  • the "first antibody” and the “second antibody” are distinct antibodies that are raised against the antigenic target of interest (for example, a protein, peptide, carbohydrate, nucleotide, deoxynucleotide, or other small molecule).
  • the second antibody binds to a different biomarker epitope than the first antibody conjugated to the biotinylated-photocleavable crosslinker, and therefore binding of the primary antibody to the biomarker does not prevent binding of the secondary antibody to the biomarker.
  • Antibodies that recognize and bind with high affinity and specificity to unique epitopes across a broad spectrum of biomolecules are available as high specificity monoclonal antibodies and/or as polyclonal antibodies.
  • antibodies are useful not only to detect specific biomolecules but also to measure changes in their level and specificity of modification by processes such as phosphorylation, methylation, or glycosylation.
  • the term "specifically binds,” referring to an antibody binding to a biomarker of choice means that the antibody binds the biomarker of choice, or subunit thereof, but does not bind to a biological molecule that is not the biomarker of choice.
  • Antibodies that specifically bind to an biomarker of choice, or subunit thereof, do not substantially cross-react with biological molecules outside the biomarker of choice.
  • the term "monoclonal antibody” includes antibodies that display a single binding specificity and affinity for a particular epitope. These antibodies are mammalian-derived antibodies, including murine, human and humanized antibodies.
  • an “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • an “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, which are generally not greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • a "portion" of a polynucleotide means at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
  • a "probe” refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions.
  • Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • an "isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • the invention includes a microfluidic purification chip, which captures at least one biomarker from an initial solution and then releases the at least one biomarker into a solution suitable for detection and quantitation.
  • the initial solution is a physiological solution.
  • the initial solution comprises blood.
  • the microfluidic purification chip may be prepared using techniques known to those skilled in the art.
  • the microfluidic purification chip may be prepared from silicon wafers according to the procedure illustrated in Figure 4.
  • the microfluidic purification chip may be prepared from moldable plastic.
  • the microfluidic purification chip may have 3D flow, i.e., can be a matrix.
  • Photoresist may be spun on the wafers and exposed using a mask.
  • the pillars used in this procedure are about 100 ⁇ m in diameter.
  • the resist pattern may be transmitted to the oxide using reactive ion etching. Resist may be stripped by ashing and the silicon pillars may be realized with an etcher.
  • the dimensions of the microfluidic purification chip determine the volume of the solution that may be contained in the chip. In one embodiment, the width of the microfluidic purification chip ranges from about 2 mm to about 6 mm. In another embodiment, the microfluidic purification chip is about 4 mm in width. In yet another embodiment, the length of the microfluidic purification chip ranges from about 5 mm to about 10 mm.
  • the microfluidic purification chip is about 7 mm in length. In yet another embodiment, the height of the microfluidic purification chip ranges from about 50 ⁇ m to about 200 ⁇ m. In yet another embodiment, the microfluidic purification chip is about 100 ⁇ m in height. In yet another embodiment, the dimensions of the microfluidic purification chip are selected so that the maximum volume of solution contained in the microfluidic purification chip is about half that of the maximum volume of solution contained in the sensing chip. In yet another embodiment, the maximum volume of solution held in the microfluidic purification chip ranges from about 1 ⁇ L to about 10 ⁇ L. In yet another embodiment, the maximum volume of solution held in the microfluidic purification chip is about 1 ⁇ L. In yet another embodiment, the maximum volume of solution held in the microfluidic purification chip is about 5 ⁇ L. In yet another embodiment, the maximum volume of solution held in the microfluidic purification chip is about 10 ⁇ L.
  • the layout and arrangement of the pillars in the microfluidic purification chip may determine the efficacy of capture of the biomarker, as illustrated in Figure 2.
  • the layout of the pillars in the microfluidic purification chip has a hexagonal geometry.
  • the layout of the pillars in the microfluidic purification chip has a triangular geometry.
  • the layout of the pillars in the microfluidic purification chip has a square geometry.
  • the number of pillar rows in the microfluidic purification chip ranges from about 10 to about 60. In yet another embodiment, the number of pillar rows in the microfluidic purification chip ranges from about 20 to about 60.
  • the number of pillar rows in the microfluidic purification chip ranges from about 20 to about 50. In yet another embodiment, the number of pillar rows in the microfluidic purification chip ranges from about 20 to about 40. In yet another embodiment, the number of pillar rows in the microfluidic purification chip ranges from about 25 to about 40. In yet another embodiment, the number of pillar rows in the microfluidic purification chip ranges from about 25 to about 35. In yet another embodiment, the number of pillar rows in the microfluidic purification chip is about 30.
  • the biomarker may be captured in the microfluidic purification chip.
  • the flow rate of the biological fluid may influence the efficiency of capture of the biomarker by the microfluidic purification chip.
  • the flow rate of biological fluid through the microfluidic purification chip ranges from about 0.1 ⁇ L/min to about 20 ⁇ L/min.
  • the flow rate of biological fluid through the microfluidic purification chip ranges from about 1 ⁇ L/min to about 15 ⁇ L/min.
  • the flow rate of biological fluid through the microfluidic purification chip ranges from about 5 ⁇ L/min to about 15 ⁇ L/min.
  • the flow rate of biological fluid through the microfluidic purification chip is about 1 ⁇ L/min.
  • the flow rate of biological fluid through the microfluidic purification chip is about 10 ⁇ L/min.
  • the microfluidic purification chip may be derivatized with a first antibody directed to the biomarker of interest so that the immobilized first antibody may capture the biomarker of interest from the biological fluid.
  • the immobilized first antibody may be attached to the surface of the microfluidic purification chip using any method known to those skilled in the art, provided that the immobilization method does not destroy the first antibody's ability to bind to the biomarker.
  • the first antibody may be attached to the microfluidic purification chip by means of a molecular crosslinker.
  • the molecular crosslinker comprises a molecular spacer and a cleavable group.
  • the cleavable group may be cleaved using a chemical reagent, such as an acid, a base, an oxidant or a reducer, or may be cleaved using a form of low-energy radiation, such as UV or visible radiation.
  • the cleavable group is a photocleavable crosslinker and may be cleaved using UV or visible radiation.
  • the photocleavable group is l-(4'-amino-2'-nitro-phenyl)ethyl.
  • the molecular spacer may be any organic molecule capable of withstanding the immobilization and release of the biomarker without undergoing significant decomposition.
  • the molecular spacer may be, for example, a peptide, a nucleic acid, a polyethylene glycol, or an alkylene group.
  • the molecular spacer is a single-stranded DNA oligonucleotide.
  • the single-stranded DNA oligonucleotide ranges in size from about 5 nucleotides to about 40 nucleotides.
  • the single-stranded DNA oligonucleotide ranges in size from about 10 nucleotides to about 30 nucleotides.
  • the single-stranded DNA oligonucleotide ranges in size from about 15 nucleotides to about 25 nucleotides.
  • the single- stranded DNA oligonucleotide is. a 20-mer. In yet another embodiment, the single- stranded DNA oligonucleotide is 5'-CGT AGA GGT TCA GTT GCA GC-3' (SEQ ID NO:3).
  • the molecular spacer may be functionalized in at least two positions. In a non-limiting example, the molecular spacer is linear and the chemical functionalities are located on opposite extremities of the spacer. In one embodiment, at least one of the chemical functionalities is coupled with a cleavable group. In another embodiment, at least one of the chemical functionalities is coupled with a photocleavable group.
  • the 1 -position of the l-(4'-amino- 2'-nitro-phenyl)ethyl photocleavable group is coupled to the molecular spacer.
  • the molecular spacer is a single-stranded DNA oligomer and the 1 -position of l-(4'-amino-2'-nitro-phenyl)ethyl is coupled to the 5 '-terminus of the single-stranded DNA oligonucleotide.
  • the cleavable group may be further coupled to a biotin-coupled moiety.
  • the 4'-amino group of the l-(4'-amino-2'-nitro- phenyl)ethyl photocleavable group is coupled to a biotin-containing moiety.
  • the molecular crosslinker is Compound (I):
  • the molecular crosslinker may be conjugated to the first antibody, using any techniques known to those in the art, with the requirement that the conjugation does not destroy the antibody's ability to bind to the biomarker of interest.
  • the molecular crosslinker has a free amino group and is coupled via an amide bond to an accessible free carboxylic group of the first antibody (from an aspartate or glutamate residue, for example).
  • the molecular crosslinker has a carboxylic group and is conjugated via an amide bond to an accessible free amino group of the first antibody (from a lysine or arginine residue, for example).
  • the amide bond formation may be performed using coupling agents such as l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N- hydroxysulfosuccinimide (NHS) in an appropriate solvent, such as an aqueous buffer or an aqueous buffer comprising water-soluble organic solvents.
  • coupling agents such as l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N- hydroxysulfosuccinimide (NHS)
  • an appropriate solvent such as an aqueous buffer or an aqueous buffer comprising water-soluble organic solvents.
  • the first antibody conjugated to the molecular crosslinker may be attached to the microfluidic purification chip through functional groups previously installed on the surface of the microfluidic purification chip.
  • the surface of the microfluidic purification chip is derivatized with a chemical group comprising avidin, and the molecular crosslinker conjugated to the first antibody comprises biotin.
  • the microfluidic purification chip may be treated with a solution of a silane, such as 3-aminopropyltriethoxysilane, under an inert atmosphere, and then heated for a defined amount of time. This treatment leads to the reaction of the silane with the surface of the chip.
  • the resulting amino groups attached to the surface of the chip may then be reacted with avidin in presence of coupling reagents such as EDC and sulfo-NHS.
  • the derivatized chip may then be treated with a solution of fetal bovine serum (FBS) to block any unreacted site.
  • FBS fetal bovine serum
  • the derivatived chip may then be treated with the first antibody conjugated to the molecular crosslinker, whereupon the biotin and the avidin moieties bind to each other and the first antibody conjugated to the molecular crosslinker becomes attached to the microfluidic purification chip.
  • the biomarker of interest may bind to the first antibody.
  • the sample may be kept in contact with the microfluidic purification chip for sufficient time to ensure appropriate binding between the biomarker and the immobilized first antibody.
  • the extent of binding of the biomarker to the immobilized first antibody may be evaluated with any method known to those skilled in the art, such as the modified ELISA test described below.
  • the solution may be drained from the chip. Washing and sensing buffers may then be perfused through the device.
  • the first antibody conjugate comprises a photolabile crosslinker
  • the microfluidic purification chip may then be charged with the buffer of choice, preferentially a buffer that does not interfere with subsequent sensing of the biomarker.
  • the captured biomarker may be released from the microfluidic purification chip by irradiating the chip with UV radiation. The length and intensity of the UV irradiation may be varied to optimize release of the captured biomarker and minimize any potential decomposition of the material. The amount of released biomarker obtained by this procedure may be evaluated by the modified ELISA test described below.
  • the microfluidic purification chip of the invention may be used for purifying the biomarker of interest from a physiological solution.
  • the microfluidic purification chip of the invention may be used for concentrating the biomarker of the interest from a physiological solution.
  • the physiological solution may be contacted with the microfluidic purification chip of the invention, to ensure that the primary antibody immobilized on the microfluidic purification chip enters in contact with the biomarker in solution and has the opportunity to capture the biomarker.
  • efficient capture of the biomarker may be achieved by prolonged contact of the microfluidic purification chip with the physiological solution comprising the biomarker.
  • efficient capture of the biomarker may be achieved by multiple contact passes of the microfluidic purification chip with the physiological solution comprising the biomarker.
  • the amount of biomarker recovered from the microfluidic purification chip may be evaluated by a modified ELISA test ( Figure 3C).
  • Figure 3C modified ELISA test
  • second antibodies to the biomarker of interest are coated on an ELISA plate, using procedures commonly known to those skilled in the art.
  • the coated plates are then washed appropriately to remove excess reagents.
  • the isolated solutions obtained by elution of the microfluidic purification chip after UV irradiation are then added to the coated ELISA plates and equilibrated.
  • the material in the wells is equilibrated with a probe comprising biotin coupled to the 3 '-terminal of a single-stranded DNA oligonucleotide that complements the single-stranded DNA oligonucleotide incorporated in the first antibody conjugate.
  • the probe is 5'-GCT GCA ACT GAA CCT CTA CGA GTG C-biotin-3' (wherein 5'- GCT GCA ACT GAA CCT CTA CGA GTG C-3' is SEQ ID NO:4).
  • the system is allowed to equilibrate in an appropriate buffer.
  • HRP streptavidin-horseradish peroxide
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the nanosensor useful within the invention is a nanoribbon sensor.
  • Such sensors may be produced from silicon-on-insulator wafers with an active and a buried oxide (BOX) layers. Such sensors may also be produced from moldable plastic.
  • the doping in the active and handle wafers is boron (p-type).
  • the nanosensors may be generated by the method discussed in the Materials section. Optical micrographs of completed devices are shown in the inset in Figures 8 and 9A.
  • the sensor may be derivatized with a second antibody directed to the biomarker of interest.
  • the second antibody binds to a different biomarker epitope than the first antibody conjugated to the biotinylated- photocleavable crosslinker.
  • the surface of the sensor may then be blocked with a protein such as FBS. Optimization of detection by the sensor may be achieved by characterizing the drain-source current (/ DS ) versus drain-source voltage (F DS ) dependence at different gate voltage (FQ).
  • FQ gate voltage
  • the region of maximum device sensitivity for the gate voltage is -2.5 V > VQ > -6 V.
  • the gate voltage VQ is about - 5 V.
  • Figure 1 schematically illustrates the operation of the microfluidic purification chip.
  • the avidin-functionalized chip ( Figure IA) is treated with antibodies to any number of specific biomarkers conjugated to biotinylated, photocleavable crosslinkers containing a specific DNA sequence.
  • Figures IB, 1C and ID illustrate the operation of the microfluidic purification chip.
  • a blood sample flows through the chip ( Figure IB) and the chip- bound antibodies bind specific soluble biomarkers, essentially purifying these molecules from whole blood.
  • wash and sensing buffers are perfused through the device.
  • Flow is then halted, and the sensing buffer-filled microfluidic purification chip is irradiated with ultraviolet (UV) light ( Figure 1C).
  • UV ultraviolet
  • Figure ID shows that after a second valve switching step transfers microfluidic purification chip contents to the nanosensor chip, the complexes bind the second antibodies on the nanowire surfaces.
  • the purification/sensing operation thus requires two specific antibody binding events for detection, a significant improvement in selectivity over previous label-free nanosensing schemes.
  • the methods described herein may be used to estimate the concentration of the biomarker of interest in the physiological solution.
  • a standard quantitative curve may be generated using the methods described herein and employing standard solutions containing known concentrations of the biomarker of interest. Such standard curve may be used to estimate the concentration of the biomarker of interest in the sensing buffer, and this concentration may be used to estimate the concentration of the biomarker of interest in the physiological solution. Any dilutions or concentrations of sample should be taken into consideration in these calculations.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • FIG. 2 shows the modeling results for the final layout.
  • a hexagonal geometry was used because previous studies demonstrated this pattern maximized capture efficiency (Nagrath et ah, 2007, Nature 450:1235).
  • Using a flow rate of 10 ⁇ L/min during biomarker binding afforded the velocity profile observed in Figure 2A (streamlines) and Figure 2B (velocities).
  • PSA prostate specific antigen
  • Chip dimensions were selected such that the volume of the microfluidic purification chip (5 ⁇ L) was equivalent to half the volume in the nanosensing reservoir, thus enabling complete transfer of microfluidic purification chip contents for sensing.
  • the microfluidic purification chip surface area can maximally bind -500 fmol of biomarker (assuming a 5 nm antibody hydrodynamic antibody radius). Complete release of bound complexes would thus produce a -100 nM biomarker solution, a value -10 6 greater than that required for any type of sensing.
  • the chip was suitable for simultaneous purification of multiple biomarkers.
  • Sl 813 photoresist was spun on the wafers and exposed using an EV Group 620 maskaligner and a mask.
  • the pillars were about 100 ⁇ m in diameter. Wafers were developed using a Hamatech-Steag automatic wafer processor. The resist pattern was then transferred to the oxide by reactive ion etching (RIE) with an Oxford Instruments PlasmaLab 80 (Oxfordshire, UK). Resist was stripped by ashing in a Branson IPC P2000 barrel etcher (San Jose, CA). Silicon pillars about 100 ⁇ m deep were realized with a Unaxis 770 Bosch etcher (St. Russia, FL) and wafers were diced into 4 mm x 7 mm chips using a K&S 7100 dicing saw (Fort Washington, PA).
  • RIE reactive ion etching
  • the photolabile crosslinker was purchased from Yale's W. M. Keck Facility (New Haven, CT) and was protected from light at all times. The sequence was 5'-Biotin-photocleavable-CGT AGA GGT TCA GTT GCA GC-amino-3', wherein 5'-CGT AGA GGT TCA GTT GCA GC- 3' is SEQ ID NO:3.
  • Antibodies to prostate specific antigen (PSA) were purchased from Accurate Chemical Co. (Westbury, NY) and antibodies to carbohydrate antigen-15.3 (CA15.3) were purchased from Alpha Diagnostics (San Antonio, TX). Antibodies were conjugated using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Pierce Scientific,
  • Chips were functionalized with 3-aminopropyltriethoxysilane (APTS) according to standard procedures (Emoto et al., 1996, Anal. Chem. 68:3751). Briefly, chips were submerged for 4 hours in a 5% (v/v) solution of APTS in anhydrous toluene in an inert nitrogen atmosphere, rinsed with toluene, and baked in a vacuum oven at 190 0 C for 12 hours.
  • APTS 3-aminopropyltriethoxysilane
  • Anti-OVA antibodies were purchased from Immunology Consultants Laboratories (Newberg, OR) and conjugated to the biotinylated photocleavable linker and bound to the microfluidic purification chip as described above.
  • Whole heparinized blood was obtained from a C56B1/6 mouse and stored at 4 0 C until use.
  • the sample was spiked with OVA-conjugated fluorescein-5- isothiocyanate (FITC) obtained from Invitrogen (Carlsbad, CA) at a concentration of 10 ⁇ g/mL. Imaging was performed with a Zeiss fluorescence microscope (Thornwood, NY). The same exposure time was used for all image captures.
  • FITC OVA-conjugated fluorescein-5- isothiocyanate
  • Percent immunoactivities were obtained by dividing pre- and post-UV irradiation concentration values determined by ELISA for each antigen. Each bar represents the average of three experiments and error bars represent one standard deviation. In order to minimize sample heating, chips were placed on ice for the duration of irradiation.
  • DIVA hybridization was performed for 2 hours at room temperature in IX SSC buffer (Promega, Madison, WI) with 0.05% sodium dodecyl suflate (SDS) and 0.1% bovine serum albumin, as described previously for solid-phase binding (Bailey et al., 2007, J. Ame. Chem. Soc. 129:1959). Washes were performed manually with IX SSC with 0.05% SDS.
  • the biotinylated sequence was 5'-GCT GCA ACT GAA CCT CTA CGA GTG C-biotin-3' and was purchased from W. M. Keck Facility (New Haven, CT).
  • Eight inch silicon-on-insulator wafers with a 70 nm active and 145 nm buried oxide (BOX) layer were purchased from SOITEC (Bernin, France) and are illustrated in Figure 7.
  • the doping in the active and handle wafers was boron (p-type) at 10 15 cm "3 .
  • the wafers were laser-cut to 4-inch diameters by Silicon Quest International (Santa Clara, CA). All photolithography steps were performed using
  • the silicon mesas were patterned in the first photolithographic (PL) step and chlorine reactive-ion etching (RIE) was performed using an Oxford PlasmaLab 100 RIE. This chemistry did not etch oxide, thus the BOX served as an etch-stop. Photoresist was stripped by ashing using a Mercator Control System Inc. HF-6 barrel asher.
  • the second PL step patterned contacted to the silicon handle wafer to serve as electronic backgates for device characterization. Vias through the BOX to the backgate were etched using 10:1 buffered oxide etch (BrandNu Labs, Meriden, CT) and photoresist was stripped using acetone and isopropanol (BrandNu Labs, Meriden, CT).
  • the third PL step patterned degenerate doping regions for contacts to device and backgate contacts.
  • a boron implant dose of 5 x 10 15 cm "2 at 8 KeV was performed at a 7° tilt by Core Systems. Photoresist was stripped by ashing, followed by wafer exposure to piranha solution. The dopant was activated by annealing the wafers at 900 0 C in nitrogen in a MRL Industries furnace after MOS cleaning.
  • the fourth PL step patterned metal leads, pads, and contacts.
  • a 75 nm Al (99.99%, Kurt J. Lesker Co.) / 75 nm Ti (99.9%, Kurt J. Lesker Co.) liftoff evaporation was performed by electron-beam deposition in a Kurt J. Lesker EJl 800 Thin Film Deposition System. After liftoff, achieved by wafer sonication in acetone, the wafers were rapid-thermal annealed (RTA) for 1 min at 650 0 C in a Surface
  • the passivation layer was deposited by PL after APTS functionalization. Devices were diced with a glass scribe and functionalized with either anti-PSA (Accurate Chemical Co.) or anti-CA15.3 (Alpha Diagnostics) using standard EDC/sulfo-NHS chemistry in IX PBS, pH 7.4. These antibodies bound different epitopes of PSA and CAl 5.3, respectively, to those conjugated to the biotinylated-photocleavable crosslinker. After washing with IX PBS, the surface was blocked with a 10% FBS solution and subsequently washed with 0.01X PBS. Reservoirs were filled with 5 ⁇ L of 0.01X PBS and remained filled with this volume until sensing measurements were performed.
  • direct measurements of the amount of the signal that would be unscreened were carried out by varying buffer salt concentration (Stern et al., 2007, Nano Lett. 7:3405).
  • the solution was then changed to a low ion concentration buffer (0.1 mM bicarbonate), which should extend the Debye length to -30 nm. The signal was observed to increase, to its maximum unscreened value.
  • a high salt concentration was then added (10 mM NaCl, ⁇ o ⁇ 3 nm), and the signal was observed to decrease far below the initial (absorbed protein) value, and close to baseline.
  • Nine devices for CA15.3 gave an average of 61% unscreened (1.6% SEM -standard error of the mean, 1 mM bicarbonate buffer).
  • Twenty PSA devices gave a slightly lower value of unscreened (46%, 2.6% SEM, 1 mM bicarbonate buffer).
  • the SPA was used in sampling mode, measuring / DS at 0.5 sec intervals, and mixing was performed with manual pipetting.
  • injection transient noise was present in all measurements (Stern et al., 2007, Nature 445:519) and devices required 1-5 mins for current stabilization in sensing buffer (Stern et al., 2007, Nano Lett. 7:3405).
  • microfluidic purification chips were fabricated from 4-inch silicon wafers in a one-step photolithographic process (Figure 4).
  • Figure 3B A scanning electron micrograph of a completed, diced chip is shown in Figure 3B. Modeling demonstrated that this geometry optimized binding ( Figure 3).
  • Chip dimensions were selected such that the volume of the microfluidic purification chip (5 ⁇ L) was equivalent to about half the volume in the nanosensing reservoir, thus enabling complete transfer of microfluidic purification chip contents for sensing.
  • the microfluidic purification chip surface area may maximally bind ⁇ 500 fmol of biomarker (assuming a 5 nm antibody hydrodynamic antibody radius).
  • the chip was thus suitable for simultaneous purification of multiple biomarkers.
  • the silicon oxide surface of the microfluidic purification chip was functionalized with 3-aminopropyltriethoxysilane (APTS) and avidin was bound using standard coupling chemistry, followed by fetal bovine serum (FBS) for blocking.
  • Antibodies were conjugated through their carboxy termini to the commercially obtained biotinylated photocleavable crosslinkers, which contained a 20-mer DNA spacer ( Figure 3A).
  • Microfluidic purification chip purification was demonstrated using anti-chicken ovatbumin (OVA) and a fluorescent protein conjugate, OVA- fluorescein-5-isothiocyanate (FITC), as illustrated in Figure 3D.
  • OVAFITC OVA- fluorescein-5-isothiocyanate
  • Figure 3D OVAFITC (10 ug/mL) was added to heparinized murine blood and flowed through the chip. After washing and flushing with sensing buffer, fluorescence imaging demonstrated specific OVAFITC binding to chip-bound antibodies ( Figure 3D).
  • PSA anti-prostate specific antigen
  • PSA is a standard clinical marker for prostate (Vickers et al., 2009, J. Clin. Oncol. 27:398-403; Shariat eta 1., 2008, Can. J. Urol. 15:4363-4374).
  • CA15.3 is a standard clinical marker for breast cancer (Rubach et al., 1997, Int. J. Biol. Markers 12:168-173; Uehara et al., 2008, Int. J. Clin. Oncol. 13:447-451).
  • Biomarker capture by microfluidic purification chips may be significantly increased by adjusting either the operation conditions, such as the flow rate into the device (modeled in Figure 2D), or the device dimensions or configuration, such as the inclusion of a recycle stream from the exit of the microfluidic purification chip.
  • nanoribbons which are devices with nanoscale thicknesses and microscale lateral dimensions (Elfstrom et al., Nano Lett. 8:945-949). These devices are less sensitive but have significant fabrication and cost advantages. Fabricated from ultra-thin silicon-on-insulator (UT-SOl) wafers using conventional lithographic techniques, these devices have been demonstrated to detect streptavidin in the 0.0318-53 ng/mL range (Elfstrom et al., 2008, Nano Lett. 8:945-949), a sensitivity range ideally suited for microfluidic purification chip-purified cancer antigen detection.
  • UT-SOl ultra-thin silicon-on-insulator
  • Antibodies were immobilized to the sensor using N-hydroxysuccinimide (NHS)/l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry.
  • NHS N-hydroxysuccinimide
  • EDC EDC
  • a buffer salt concentration varying buffer salt concentration. This study indicated that -50% of the signal was not screened by the buffer solution.
  • the normalized responses of anti-PSA and anti-CA15.3 functionalized devices to this solution are illustrated in Figures 14A and 14B, respectively, and demonstrate successful detection of these antigens.
  • the asymptotic saturation value of the device response is weakly dependent on concentration for reversible reactions with a low dissociation constant, which is the case for the antigen-antibody interactions.
  • the initial kinetic reaction rates were selected instead of endpoint detection.
  • the concentrations matched those used for direct sensing buffer measurements ( Figures 9E-F), thus similar device responses were anticipated. Indeed, similar signals were obtained, demonstrating effective, consistent, and integrated microfluidic purification chip operation.

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Abstract

La présente invention porte sur une puce microfluidique de purification pour capturer un biomarqueur à partir d'une solution physiologique. La présente invention porte également sur un procédé de capture et de libération d'un biomarqueur, ce dernier étant initialement dans une solution physiologique. La présente invention porte en outre sur un procédé de pré-purification et de mesure de la concentration d'un biomarqueur dans une solution physiologique.
PCT/US2010/025412 2009-02-27 2010-02-25 Préparation d'échantillon physiologique pour nanodétecteurs WO2010099318A1 (fr)

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US9599614B2 (en) 2011-03-14 2017-03-21 Yale University Calibration of nanostructure sensors
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US9316612B2 (en) 2013-01-04 2016-04-19 Yale University Regenerative nanosensor devices

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